Solubility enhancement of berberine–baicalin complex by the constituents of Gardenia Fruit

A Kampo prescription usually consists of several crude drugs and contains many kinds of compounds. Physicochemical interactions between the compounds may occur in the process of decoction, by which Kampo prescriptions are usually prepared for ingestion, and the interactions may change the extraction yields of the constituents. Berberine and baicalin have been reported to form precipitates. Orengedokuto, which consist of Coptis Rhizome, Gardenia Fruit, Phellodendron Bark and Scutellaria Root, has been a representative Kampo prescription used to treat inflammatory diseases. In our previous papers, we revealed that the precipitates formed in the decoction of orengedokuto without Gardenia Fruit mainly consists of berberine–baicalin complex and that Gardenia Fruit reduced the amount of the precipitates in orengedokuto decoction. In this report, through solubility-enhancement assay based on HPLC, we identified crocins as the constituents of Gardenia Fruits, which enhanced the solubility of berberine–baicalin complex. All-trans crocin-1 (1) and 13-cis crocin-1 (5) showed high activities among the isolated crocins, and the number of glucosyl groups in the molecule seemed correlated with the activity. As berberine and baicalin were reported as the anti-inflammatory constituents of Coptis Rhizome and Phellodendron Bark, and Scutellaria Root, respectively, Gardenia Fruit contributes anti-inflammatory activity of orengedokuto by increasing solubilities of anti-inflammatory constituents of the other component crude drugs in the prescription. Our result will add a scientific basis to the understanding of the effectiveness of orengedokuto as a whole.


Introduction
A Kampo prescription usually consists of several crude drugs and contains many kinds of compounds. Physicochemical interactions between the compounds may occur in the process of decoction, by which Kampo prescriptions are usually prepared for ingestion, and the interactions may change the extraction yields of the constituents [1,2]. Precipitate formation is one of the interactions causing such changes [3]. Interactions of berberine with baicalin [4], glycyrrhizin [5], tannins [3], and wogonoside [6] are typical examples of such precipitate formation out from a decoction.
Orengedokuto (黄連解毒湯, huanglianjiedutang), consisting of Coptis Rhizome, Gardenia Fruit, Phellodendron Bark and Scutellaria Root, contains berberine, a characteristic component of Coptis Rhizome and Phellodendron Bark, and baicalin, the major constituent of Scutellaria Root. Recent study showed that the yellow precipitates formed in decoction of orengedokuto were mainly composed of berberine and baicalin [4], and our group revealed that the constituents of Gardenia Fruit reduced the amount of berberine-baicalin precipitates in orengedokuto decoction [7]. Gardenia Fruit, the fruit of Gardenia jasminoides Ellis (Rubiaceae), is a crude drug used in Asian traditional medicines. Anti-hypertension [8], antioxidant [9], anxiolytic [10], choleretic [11], and neuroprotective [12] activities have been reported for Gardenia Fruit. Representative constituents of Gardenia Fruit are iridoid glycosides (geniposide, genipin gentiobioside, etc.) [13], crocins [14], monoterpenoids (jasminosides and sacranosides) [15], and quinic acid derivatives [16]. However, there has been no report on the compound responsible for the solubility-enhancement of the berberine-baicalin complex. In this paper, we describe the identification of the compounds in Gardenia Fruit involved in solubility-enhancement of berberine-baicalin precipitates.

HPLC based solubility assay
Berberine hydrochloride was dissolved in water at 60 ºC to prepare 2 mM solution. Baicalin was dissolved in methanol to prepare 4 mM solution. The assay samples were dissolved in dimethyl sulfoxide (DMSO) and diluted 100 times with water to adjust their concentration at 800 µg/mL (1% DMSO). Solutions of berberine hydrochloride (200 µL), baicalin (100 µL), and a sample (500 µL) were added to a 1.5 mL-Eppendorf tube in this order, mixed for 30 s with VORTEX-GENIE 2 Mixer (M&S Instruments Inc., Osaka, Japan), and left for 60 min at room temperature. The mixture was centrifuged at 13,000g for 10 min, and then 100 µL of the supernatant was concentrated to dryness under reduced pressure. The residue was dissolved in 100 µL of methanol and analyzed by HPLC. One percent of DMSO was used as a control solution.
As a measure of solubility-enhancement activity, relative solubilities of berberine and baicalin in an assay mixture compared to the control solution (without any sample) were calculated from their peak areas of HPLC chromatogram monitored at UV 275 nm by the following equation: R e l a t i v e s o l u b i l i t y (%) = peak area of berberine or baicalin in an assay peak area of berberine or baicalin in the control (DMSO) × 100 (%).

Statistical analysis
The significant differences in the relative solubilities between the control and the assay samples were tested by Dunnett's multiple comparison test using R statistical software. The results of the relative solubility of berberine and baicalin were analyzed statistically by a two-tailed Student's t test.

Identification of the constituents with solubility-enhancement activity from Gardenia Fruit
To isolate compounds that enhance the solubility of berberine-baicalin complex from Gardenia Fruit, activity-guided fractionation was conducted. Gardenia Fruit was extracted with hot water, then the extract was fractionated by a Diaion HP-20 column eluted with water (DW), 20% MeOH (DM-1), 50% MeOH (DM-2), MeOH (DM-3) and acetone (DA). The solubility-enhancement activity of each fraction was evaluated by HPLC based solubility assay (Fig. 3). Fraction DM-3 showed significant solubility-enhancement (the relative solubilities of berberine and baicalin at 500 µg/mL were 172% and 191%, respectively). Similarly, DM-2 and DA also showed the activity though the potency was lower than in the DM-3. In TLC analyses of these fractions, yellow spots were detected in DM-2, DM-3, and DA, whereas no yellow spot was observed in both DW and DM-1. These yellow spots corresponded to those detected in commercially available crocin (Gardenia Fruit extract). These results suggested that solubility-enhancement activity observed in DM-2, DM-3 and DA was attributable to crocins.
Next, to evaluate the solubility-enhancement activity of crocins, the activity of commercially available crocin was measured at various concentrations (Fig. 4). It showed concentration-dependent activity, and the relative solubilities of berberine and baicalin at 500 µg/mL were 179% and 211%, respectively. These results indicated that crocins contained in Gardenia Fruit were responsible for the solubilityenhancement activity to the baicalin-berberine complex.

Solubility-enhancement activities of crocins and their mixture
To evaluate the solubility-enhancement activity of isolated crocins 1-5, their relative solubilities for berberine and baicalin in the presence of 1-5 alone and also of their mixture were measured (Fig. 6). Three compounds (1, 3, and 5) and the mixture, whose ratio of 1-5 was identical to that found in the commercially available crocin, showed high activity for berberine (about 200%) and baicalin (about 235%), whereas the activities of compounds 2 and 4 were lower. Compounds 1, 3 and 5 showed the same level of activity at 500 µg/mL. However, as the molecular weights of 1 and 5 (MW 976) are larger than that of 3 (MW 652), the activities of 1 and 5 in molar concentration basis were higher than that of 3.
The mixture of 1-5 showed the same level of solubility-enhancement with 1 and 5. This result suggested that these compounds act in the same mechanism for solubilityenhancement. The commercially available crocin (positive control), whose HPLC chromatogram implied the presence of compounds other than crocins (Fig. 2), showed lower activity than the crocin mixture (Fig. 6). The relative solubilities of commercially available crocin were 164% and 202%, and those of the crocin mixture were 200% and 246%, for berberine and baicalin, respectively. As the total content of crocins in the commercially available crocin was about 80% expected from the HPLC chromatogram (Fig. 2b), these results seemed well corresponded. Compounds 1, 3, 5 and the crocin mixture showed significantly higher activity to baicalin than berberine, indicating that solubility of baicalin was more enhanced by these crocins than that of berberine.

Discussion
Orengedokuto has been a representative prescription for heat-clearing and detoxicating and used to treat inflammatory diseases [21]. In a previous paper, we showed that berberine, the constituent of Coptis Rhizome and Phellodendron Bark, contributed to the anti-inflammatory activity of orengedokuto through inhibition of nitric oxide production [7]. We also revealed that flavonoids of Scutellaria Root such as baicalein and wogonin played a central role in the inhibition of prostaglandin production by orengedokuto [22,23]. These flavonoids are mainly contained as glycosides in Scutellaria Root and absorbed as themselves and as their aglycon [24]. Berberine forms a complex with baicalin and wogonoside, the major flavone glycosides of Scutellaria Root whose aglycon are baicalein and wogonin, respectively, and precipitates out from aqueous solution [4,6]. This means that the anti-inflammatory constituents of orengedokuto would be reduced in the process of decoction, leading to be less effectiveness. In fact, a large amount of precipitates, which mainly consisted of berberine and baicalin, was observed in a decoction prepared from Coptis Rhizome, Phellodendron Bark and Scutellaria Root. However, the amount of the precipitates was largely decreased in a decoction of orengedokuto, which contained Gardenia Fruit in addition to the mixture [7]. In this study, through activityguided fractionation, we identified crocins as the compounds in Gardenia Fruit involved in the solubility-enhancement of berberine-baicalin complex. Thus, Gardenia Fruit contributes anti-inflammatory activity of orengedokuto by increasing solubilities of anti-inflammatory constituents of the other component crude drugs in the prescription. In our study, we focused on anti-inflammatory activity of orengedokuto. However, other pharmacological activities of berberine and baicalin have been reported. Berberine inhibits α-glucosidase [25], exhibits antidiarrheal effect [26], and has antibacterial activity [27], and baicalin shows antithrombotic activity [28] etc. Therefore, Gardenia Fruit may also be involved in other therapeutic effects of orengedokuto. Thus, our result will add scientific basis to the understanding of the effectiveness of orengedokuto as a whole.
Kampo prescriptions and their effectiveness have been established through many experiences of applications to patients. However, the scientific basis of the effectiveness of Kampo prescriptions, especially how interactions among constituents contribute to the effect of a Kampo prescription, is still limited. Further investigations based on the Fig. 6 Solubility-enhancement of berberine-baicalin complex by compounds 1-5 and their mixture at 500 µg/mL The results are expressed as the mean ± SD of three independent experiments. An asterisk indicates a significant difference with control (p < 0.01). A dagger indicates a significant difference between baicalin and berberine (p < 0.05). cont.: without sample, pos.: commercially available crocin (500 µg/mL) constituents to understand the effectiveness of a Kampo prescriptions will be necessary.
Berberine and baicalin are reported to form a 1:1 complex [4,6], and Wang et al. reported that carboxyl group of baicalin and the quaternary ammonium ion of berberine are facing inside, and hydrophobic parts of baicalin and berberine are outside in the complex [6]. As the crocins with solubilityenhancement activity have a large hydrophobic polyene moiety and hydrophilic glycoside parts on both sides, and the activity seemed correlated with the number of sugars, they may act as a surfactant to dissolve the complex. However, as the relative solubilities of berberine and baicalin are different (Fig. 6), crocins may act differently to berberine and baicalin. Investigations on the mechanism of the solubilityenhancement of crocins are in progress in our laboratory and will be discussed in a forthcoming report. In the highly polar fractions [DW and DM-1 (Fig. 3), C1 (Fig. 5)], the solubility of berberine was lowered while that of baicalin was the same level with the control. This suggested the presence of compounds which specifically interact with berberine in these fractions. Further investigation will be necessary to identify the compounds.